The present invention relates to recombinant microorganisms, in particular gut-colonising organisms, which are useful for example in the delivery of antigenic material and thus form the basis of vaccines. Vaccines comprising these organisms and promoter sequences for use in them form a further aspect of the invention.
Attenuated mutants of Salmonella typhi (e.g. aroA, aroC, htrA) are currently being evaluated as live, oral vaccines against typhoid fever (Tacket C O, et al., Infect. Immun. 1997;65:452-6). These mutants have also attracted attention as carriers for guest (vaccine) antigens but suitable animal models for testing these vaccines are not available. In view of this, many workers have used Salmonella typhimurium aroA expressing guest antigens for investigating the immune responses induced after oral vaccination of mice.
The unregulated expression of foreign genes within Salmonella species such as S. typhimurium can lead to plasmid instability, yet the stable expression of the guest antigen at the appropriate site in the body is necessary for the induction of a protective response. One approach to promote the stable expression of guest antigens involves the chromosomal integration of the heterologous gene. However, this may reduce the immune response because of gene dosage effects (Covone M G, et al., Infect. Immun. 1998;66:224-31).
The balanced lethal system (Curtiss R III, et al., Res. Microbiol. 1990;141:797-805, Nakayama K, et al., Bio/Technology 1988;6:693-97) relies on the complementation of a lethal mutation by a plasmid which also encodes the guest antigen. Whilst this ensures retention of the plasmid, the gene encoding the guest antigen itself may be deleted. An alternative approach involves the use of promoters which are induced within host tissues to direct guest antigen expression at that site. Because the gene is only expressed after certain environmental cues have been recognised, this approach might reduce the selective pressure towards deleting the gene.
This solution to the problem of expression of guest antigens has also been identified by other workers. A variety of antigens have been expressed in S. typhimurium from the nirB promoter which is upregulated under anaerobic conditions and within host cells (Oxer M D, et al. Nucleic Acids Res. 1991;19:2889-92). Guest antigens delivered using the nirB promoter system induce superior responses than the same antigens delivered from a constitutive promoter. In addition, the nirB promoter-driven genes were maintained more effectively in the Salmonella host strain. More recently, it has been shown that the htrA and osmC promoter can be used to direct expression of guest antigens in Salmonella (McSorley S J, et al., Infect. Immun. 1997;65:171-78, Roberts M, et al., Infect. Immun. 1998;66:3080-87). However, it is likely that these promoters will not be suited to the expression of all guest antigens.
Immunisation with the F1-antigen of Y. pestis has previously been shown to induce an antibody-mediated protective response against plague (Green M, et al., FEMS Microbiology and Immunology, 1998;23:107-13) and we have previously shown that the F1-antigen can be expressed in S. typhimurium (Oyston P C F, et al., Infect. Immun. 1995;63:563-68, Titball R W, et al., Infect. Immum. 1997;65:1926-30). The antigenic properties of F1-antigen have been exploited to investigate the ways in which different promoters, which are induced at different sites in the body, can be used to induce different antibody responses to guest antigens expressed in S. typhimurium. It is known that the invasion and spread of S. typhimurium within the host is accompanied by the expression of different subsets of genes which are involved in processes such as attachment and invasion, penetration of the epithelium and the infection of deep lymphoid tissue.
The OmpR/EnvZ two component regulatory system responds to changes in the osmotic strength and pH within S. typhimurium (Foster J W, et al., Microbiology 1994;140:341-52). It has been suggested that this system might play a role in allowing the bacterium to survive in the gut by regulating the expression of outer membrane porins such as OmpC (Pratt L A, et al., American Society of Microbiology, ASM Press, Washington D.C., 1995, pp105-27, Nikaido H, et al., Cellular and Molecular Biology. American Society for Microbiology, Washington D.C. 1987, p7-22, Garcia Véscovi E. et al., Cell. 1996;84:165-74).
The PhoP/PhoQ two-component regulatory system controls virulence properties such as survival within macrophages, resistance to host defence antimicrobial peptides and acid pH, invasion of epithelial cells, the formation of spacious vacuoles and the processing and presentation of antigens by activated macrophages (Miller S I. et al., Proc. Natl. Acad, Sci USA 1989;86:5054-58, Fields P I, et al., Science 1989;243:1059-62, Pegues D A, et al., Mol. Microbiol. 1995;17:169-81, Wick M J, et al., Mol. Microbiol. 1995;16:465-76), in response to environmental magnesium concentration (García Véscovi E. et al., Cell. 1996;84:165-74). Over forty genes are regulated by this system in S. typhimurium (Soncini F C, et al., J. Bacteriol. 1996;178:5092-99) including the phoP gene, which is autoregulated (Soncini F C, et al., J. Bacteriol. 1995;177:4364-71) and the pagC gene which encodes an envelope protein required for survival in the macrophage (Alpuche-Aranda C M, et al., Proc. Natl. Acad. Sci. USA 1992;89:10079-83).
Attenuation of Salmonella by partial deletion of the pagC gene and fusion to a heterologous protein is described in U.S. Pat. No. 5,733,760.
The applicants have however found that certain promoters can be used advantageously in such systems to drive high levels of expression of heterologous proteins, in particular in mucosal cells.